Sorghum is a short-day monocotyledon of the Poaceae family originating in Africa. Sorghum has a high water use efficiency, as well as excellent drought resistance and high-temperature resistance, and it is a major cereal crop next to corn, rice, wheat and barley, constituting an important food supply in African and Asian countries. Because Sorghum has a high sugar content and can be a raw material for bioethanol, much effort is put into technological developments to modify the agricultural characteristics of Sorghum. The genome sequencing of Sorghum has been completed, and an analysis of genes involved in the useful agricultural characteristics specific to Sorghum as well as of the quantitative trait locus (QTL) is in progress. The technological development of Sorghum gene recombination is considered a crucial factor in modifying the agricultural characteristics of Sorghum. 
Physicochemical methods (direct introduction of DNA), such as a polyethylene glycol method, an electroporation method, and a particle gun method, and biological methods (indirect introduction of DNA) utilizing functions of Agrobacterium are known as methods for transformation of monocotyledons such as barley, wheat, corn, and rice, which are major cereal crops. In the gene introduction mediated by Agrobacterium (hereinafter referred to as the Agrobacterium method), the regulation of expression of gene groups in a Ti plasmid virulence region (vir region) maintains a small number of copies of an objective gene and prevents a gene from being introduced as fragmented segments. The gene introduction mediated by Agrobacterium therefore has notable advantages of providing a large number of transformants highly expressing the objective gene and of allowing the difference in expression levels among individual transformants to be small compared to the direct gene introduction.
The Agrobacterium method is universally used for transformation of dicotyledons. Although it has been believed for a long time that hosts of Agrobacterium in nature are limited only to dicotyledons and that Agrobacterium has no ability to infect monocotyledons, a method of a highly efficient transformation of a monocotyledon by Agrobacterium has been reported, the first of which was in a major cereal crop, rice, as a result of detailed studies such as investigation of tissue materials, improvements in medium compositions, and selection of Agrobacterium strains (Hiei et al., 1994: Non-Patent Document 1). Then, following on the success in rice, examples of successful transformation mediated by Agrobacterium in corn (Ishida et al., 1996: Non-Patent Document 2), wheat (Cheng et al., 1997: Non-Patent Document 3), barley (Tingay et al., 1997: Non-Patent Document 4), and Sorghum (Zhao et al., 2000: Non-Patent Document 5) have been reported.
1. Known Technologies for Transformation of Sorghum by the Agrobacterium Method
1) Plant Tissue Inoculated with Agrobacterium 
An immature embryo is used as the tissue of Sorghum to be inoculated with Agrobacterium (Non-Patent Document 5).
2) Pre-Treatment
A thermal treatment or a centrifugation treatment of the plant material to be transformed is used in several examples of transformation to a monocotyledon plant as a method to improve the transformation efficiency of the Agrobacterium method (Hiei et al. 2006: Non-Patent Document 6). It has been reported that the transformation efficiency of Sorghum also increased when immature embryos were treated at 43° C. for 3 min before infection by Agrobacterium, so that the transformation efficiency of the treated plot increased by about three folds relative to a non-treated plot (Gurel et al. 2009: Non-Patent Document 7). With regard to the centrifugation treatment, however, it is written that the treatment had a negative effect on the transformation of Sorghum. 
3) Co-Cultivation Step
This step is a step of inoculating a plant tissue with Agrobacterium to co-cultivate the plant tissue and Agrobacterium. MS medium which contains a one-time amount of MS Inorganic Salts as the composition of a co-cultivation medium is used in the transformation of Sorghum mediated by Agrobacterium (Non-Patent Document 5).
4) Callus Formation Step
The callus formation step is a step for dedifferentiating plant tissues.
Callus formation tends to be inhibited in the Sorghum tissue culturing step due to excessive generation of phenol compounds (Cai et al. 1987: Non-Patent Document 8). Meanwhile, it has been reported that an addition of L-proline and L-asparagine to the MS medium accelerated the growth of embryogenic callus and suppressed the generation of phenol compounds which constitutes an inhibitory cause against culturing Sorghum (Elkonin et al. 1995: Non-Patent Document 9). It has also been reported that an increased nitrate ion concentration and phosphate ion concentration promoted the induction of embryogenic callus and improved the regeneration efficiency from callus to a plant (Elkonin et al. 2000: Non-Patent Document 10). It is also reported that an addition of polyvinylpolypyrrolidone (PVPP) to a callus formation medium reduces the browning of cells from damages that occurred when the cell was inoculated with Agrobacterium (Gao et al. 2005: Non-Patent Document 11).
As an exemplary case of an actual Sorghum transformation, there is a report that the transformation efficiency improved by using 6-benzylaminoproline (BAP) as the plant growth regulator to be added to the callus formation medium (resting medium and selection medium) (Wu et al. 2014: Non-Patent Document 12).
5) Regeneration Step
There are attempts to optimize the types and concentrations of plant growth regulators to be added to the regeneration medium. It has been reported that regeneration of roots from the regenerated plant transplanted to a rooting medium is promoted by using MS medium that includes 1 mg/L naphthaleneacetic acid (NAA), 1 mg/L indole-3-acetic acid (IAA), 1 mg/L indole-3-butyric acid (IBA) and 1 μmol/L copper sulfate (Guoquan et al. 2013: Non-Patent Document 13).
The transformation method of Sorghum mediated by Agrobacterium has been modified as shown above, and the transformation efficiency that was 2.1% (Non-Patent Document 5) in the first report increased to 33% (Non-Patent Document 12). However, this transformation efficiency is still not high enough relative to rice or corn.
2. Modification of Co-Cultivation Medium in Plant Transformation Mediated by Agrobacterium Bacteria
Multiple reports have been provided in relation to corn regarding transformation mediated by Agrobacterium of an improvement in transformation efficiency by the use of a co-cultivation medium consisting of a diluted N6 medium. For example, Du et al. showed that among corn (A188) immature embryos inoculated with Agrobacterium, the rate of immature embryos exhibiting transient expression of GUS genes increased to 90% or higher for all N6 mediums whose main inorganic salts were diluted to 50%, 30% or 10% relative to 72% in N6 medium containing N6 Inorganic Salts at a one-time amount (Du et al. 2010: Non-Patent Document 14).
Furthermore, another report on corn (Hi-II) demonstrates that while a use of an Agrobacterium suspension medium and a co-cultivation medium each containing N6 Inorganic Salts at a one-time amount resulted in 0% transformation efficiency per contesting callus, a use of an Agrobacterium suspension medium and a co-cultivation medium each containing N6 main Inorganic Salts diluted to 10%, 50% increased the transformation efficiencies to 4.0% and 17.5%, respectively (Vega et al. 2008: Non-Patent Document 15).
It is reported concerning wheat that a use of an Agrobacterium suspension medium and a co-cultivation medium each consisting of MS medium comprising inorganic salts diluted to 10% provided a significantly high rate of immature embryos exhibiting transient expression of GUS genes than the above medium consisting of a one-time amount of MS Inorganic Salts (Cheng et al. 1997: Non-Patent Document 16). As such, it is considered that “low inorganic salt mediums are generally used to improve the transient expression or transformation efficiency of the gene introduced to several major crops by Agrobacterium (Vega et al. 2008: Non-Patent Document 15).”
On the other hand, although not a co-cultivation medium, a use of a medium having an increased ammonium nitrate concentration as a pre-culture medium for the plant material of tobacco before inoculation with Agrobacterium was reported to have increased the transient expression of the introduced gene and the transformation efficiency (Boyko et al. 2009: Non-Patent Document 17). It is also reported that the transformation efficiency of Arabidopsis and tobacco increased by an addition of potassium chloride or a chloride of rare earth elements to the medium for pre-culture of plant tissues before inoculation with Agrobacterium (Boyko et al. 2011: Non-Patent Document 18).
There is also report of promoting a transient expression of introduced genes by adding antioxidant materials or copper to a co-cultivation medium. With regards to corn, it was reported that the transient expression of introduced gene and the transformation efficiency increased significantly when L-cysteine (0.4 g/L) and dithiothreitol (DTT) (0.4 g/L), which are anti-oxidant materials, were added to the MS medium, N6 medium, LS medium, and the D medium, relative to a plot where none was added (Non-Patent Document 14).
3. Medium Used in Plant Tissue Culture
In addition to the above MS medium (LS medium), mediums of various constitution are developed and used in culturing plant tissues.
The N6 medium was used mainly as another culture medium of rice. The B5 medium was developed for use in liquid culture, but it is also used as a solid medium in many plant cell cultures. The R2 medium is a medium based on the B5 medium and developed for use in suspension culture cells of rice. The CC medium was devised as a medium for forming callus from the protoplast of corn.
The following chart shows the total concentration of NH4+ and NO3− as the nitrogen source and the concentrations of various inorganic ions (magnesium ion, potassium ion, calcium ion, and sodium ion) in the MS medium, LS medium, N6 medium, B5 medium, R2 medium and the CC medium.
TABLE 1MSLSN6B5R2CCNH4+, NO3− Total (mM)60.060.035.027.045.028.0mg2+ (mM)1.51.50.82.01.01.0K+ (mM)20.020.030.925.040.013.0Ca2+ (mM)3.03.01.11.01.04.0Na+ (mM)0.00.00.01.12.00.0